The objective of the proposed research is to characterize the tobacco-mediated inhibition and inactivation of NO-synthase (NOS), a cytochrome P450-like enzyme. In the course of these studies, we aim to isolate and identify the chemicals in tobacco that are responsible for these effects. This will be addressed, in part, by a differential metabolic profiling approach, as well as the use of high-throughput bioassays and state of the art LC-MS/MS techniques. Exposure to chemicals in tobacco is known to cause a dysfunction in NO production and availability. Cigarette smoking is associated with a variety of diseases and, since NO plays a central role in a myriad of physiological functions, it is likely that such interactions may be clinically important. We have established, with the use of crude extracts derived from tobacco and tobacco smoke, that there is an immediate, reversible inhibition of NOS, as well as a metabolism-based, time-dependent, irreversible inactivation of NOS. Further studies on the extracts indicate that small, organic, water-soluble compounds are responsible for causing NOS dysfunction. Moreover, these extracts specifically inactivated endothelial NOS (eNOS) when L-arginine was present, whereas specific inactivation of neuronal NOS (nNOS) was observed when tetrahydrobiopterin was in excess. The following specific aims are proposed: 1. To characterize the inactivation of nNOS caused by tobacco components. This includes identification of the chemicals responsible for inactivation. 2. To characterize the inactivation of eNOS caused by tobacco components. The mechanism of tetrahydrobiopterin oxidation, as well as the identity of the chemicals responsible for inactivation will be addressed. 3. To characterize the mechanism of inhibition of eNOS and nNOS caused by tobacco smoke, as well as determine the identity of the components responsible. The successful completion of these aims will identify potentially novel inhibitors and inactivators of NOS as pharmacological tools, as well as allow for a more detailed study of the effects of these chemicals in smokers. Since over 40 million people are exposed to these chemicals on a daily basis, information on the nature of these chemicals and their interaction with important biological systems would be useful. These studies should also lead to identification of biomarkers for tobacco-related diseases involving NOS and facilitate development of therapies for NOS dysfunction.